• YAKHAK HOEJI
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• v.47 no.1
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• pp.31-36
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• 2003

To investigate the effect of protein kinase on melanin production via cAMP-dependent pathway, we measured the melanin amount and tyrosinase activity in B16 melanoma cells stimulated by alpha-melanocyte stimulating hormone (MSH), forskolin and 8-Br-cAMP. MSH, forskolin and 8-Br-cAMP significantly increased both melanin production and tyrosinase activity in B16 cells. Melanin production and tyrosinase activity by MSH are significantly inhibited by cyclic AMP-dependent protein kinase inhibitor (KT5720) and protein kinase C down-regulation treated with PMA. Bisindolmaleimide (1$\mu$M), protein kinase C inhibitor, significantly inhibited melanin production and tyrosinase activity stimulated by MSH, forskolin and 8-Br-cAMP with the following order of potency: MSH＞forskolin＞8-Br-cAMP. Tyrosine kinase inhibitor, genistein and DHC, significantly inhibited both, but the inhibitory effect was more potent in 8-Br-cAMP-stimulated B16 cells than MSH-stimulated cells. NFkB inhibitor (parthenolide) significantly inhibited melanin production and tyrosinase activity. Neither melanin production nor tyrosinase activity induced by MSH, forskolin and 8-Br-cAMP were affected by KN-62 (calmodulin-dependent protein kinase II inhibitor), PD098059 (mitogen-activated protein kinase inhibitor, MAPKK) and worthmannin (phosphatidylinositol 3-kinase inhibitor). These results suggest that both protein kinase C and tyrosine kinase are involved in melanin production by cyclic AMP-dependent pathway and NFkB pathway may play an important role in cyclic AMP-dependent melanin production in B16 melanoma cells.

• YAKHAK HOEJI
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• v.37 no.6
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• pp.598-604
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• 1993

The calyx extract of Campsis grandiflora displayed inhibitory activity against protein kinase C from the bovine brain. Separation guided by protein kinase C enzyme assay and bleb forming assay led to isolation of a potent protein kinase C inhibitor that was identified as a known phenylpropanoid glycoside, verbascoside. It suppressed completely bleb-formation of K562 cell surface induced by phorbol 12,13-dibutylate at the concentration of 60 $\mu\textrm{g}$/ml and IC$_{50}$ of the protein kinase C occured at 20 $\mu{M}$. This compound was tested for cytotoxic activity against ten human tumor cell lines in vitro. it exhibited moderate cytotoxic activity against skin tumor cell line M14 (IC$_{50}$ 2.2 $\mu\textrm{g}$/ml) and very weak cytotoxicity against other cell lines (IC$_{50}$>10 $\mu\textrm{g}$/ml)

• Archives of Pharmacal Research
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• v.21 no.3
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• pp.315-319
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• 1998

Gastric smooth muscle of cats was used to investigate the involvement of protein kinase in vanadate-induced contraction. Vanadate caused a contraction of cat gastric smooth muscle in a dose-dependent manner. Vanadate-induced contraction was totally inhibited by 2 mM EGTA and 1.5 mM $LACI_3$ and significantly inhibited by $10\mu$M verapamil and $1\mu$M nifedipine, suggesting that vanadate-induced contraction is dependent on the extracellular $Ca^{2+}$ concentration, and the influx of extracellular $Ca^{2+}$ was mediated through voltage-dependent $Ca^{2+}$ channel. Both protein kinase C inhibitor and tyrosine kinase inhibitor significantly inhibited the vanadate-induced contraction and the combined inhibitory effect of two protein kinase inhibitors was greater than that of each one. But calmodulin antagonists did not have any influence on the vanadate-induced contraction. On the other hand, both forskolin ($1\mu$M) and sodium nitroprusside ($1\mu$M) significantly inhibited vanadate-induced contraction. Therefore, these results suggest that both protein kinase C and tyrosino kinase are involved in the vanadate-induced contraction which required the influx of extracellular $Ca^{2+}$ in cat gastric smooth muscle, and that the contractile mechanism of vanadate may be different from that of agonist binding to its specific receptor.

• YAKHAK HOEJI
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• v.44 no.2
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• pp.162-168
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• 2000

To investigate the effects of protein kinases on endothelin-1-induced phospholipase C activation and $Ca^{2+}$ mobilization in Rat-2 fibroblast, we measured the formation of inositol phosphates and intracellular $Ca^{2+}$ concentration with [$^3$H]inositol and Fura-2/AM, respectively. Endothelin-1 dose-dependently activated phospholipase C and increased intracellular $Ca^{2+}$ concentration. Protein kinase C activator PMA, significantly inhibited both phospholipase C activity and $Ca^{2+}$ mobilization induced by endothelin-1. Tyrosine kinase inhibitor, genistein, inhibited both. On the other hand, cyclic nucleotide (cAMP and cGMP) did not have any influence on the signaling pathway of phospholipase C-Ca$^{2+}$ mobilization induced by endothelin-1. These results suggest that protein kinase C and tyrosine kinase counteract on the signaling pathway of phospholipase C-Ca$^{2+}$ mobilization induced by endothelin-1 in Rat-2 fibroblast. fibroblast.

• Proceedings of the Zoological Society Korea Conference
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• 1998.04a
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• pp.56.1-56
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• 1998

No Abstract, See Full Text

• Tuberculosis and Respiratory Diseases
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• v.44 no.2
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• pp.338-348
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• 1997

Background : Endotoxin, the component of outermembrane of gram negative organism, plays an important role in the initiation and amplification of inflammatory reaction by its effects on inflammatory cells. Until recently, there have been continuing efforts to delinate the mechanisms of the signal trasduction pathway of endotoxin stimuli on inflammatory cells. By uncovering the mechanisms of signal transduction pathway of endotoxin stimuli, we can expect to have tools to control the excessive inflammatory responses which sometimes may be fatal to the involved host. It was generally accepted that endotoxin exerts its inflammatory effects through inflammatory cytokines that are produced by endotoxin-stimulated inflammatory cells and there were some reports on the importance of protein kinase C and protein tyrosine kinase activation in the production of inflammatory cytokines by endotoxin So we evaluated the effect of pretreatment of protein kinase C inhibitors (H7, Staurosporin) and protein tyrosine kinase inhibitors(Herbimycin, Genistein) on the endotoxin-stimulated cytokines(IL-8 & TNF-$\alpha$) mRNA expression. Method : Peripheral blood monocytes were isolated from healthy volunteers by Ficoll-Hypaque density gradient method and purified by adhesion to 60mm Petri dishes. Endotoxin(LPS 100ng/ml) was added to each dishes except one control dish, and each endotoxin-stimulated dishes was preincubated with H7, Staurosporin(protein kinase C inhibitor), Herbimycin or Genistein(protein tyrosine kinase inhibitor) respectively except one dish. Four hours later the endotoxin stimulation, total RNA was extracted and Northern blot analysis for IL-8 mRNA and TNF-$\alpha$ mRNA was done. Result : Endotoxin stimulation increased the expression of IL-8 mRNA and TNF-$\alpha$ mRNA expression in human peripheral blood monocyte as expected and the stimulatory effect of endotoxin on TNF-$\alpha$ mRNA expression was inhibited by protein kinase C inhibitors(H7, Staurosporin) and protein tyrosine kinase inhibitors (Herbimycin, Genistein). The inhibitory effect of each drugs was increased with increasing concentration. The stimulatory effect of endotoxin on IL-8 mRNA was also inhibited by H7 and protein tyrosine kinase inhibitors (Herbimycin, Genistein) dose-dependently but not by Staurosporin. Conclusion : Protein kinase C and protein tyrosine kinase are involved in the endotoxin induced signal transduction pathway in human peripheral blood monocyte.

• BMB Reports
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• v.34 no.6
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• pp.517-525
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• 2001

Six renaturable protein kinases that utilize the myelin basic protein (MBP) as a substrate were activated during prolonged exposure of cardiac myocytes to okadaic acid (OA). We characterized the substrate preference and activation of these kinases, with particular emphasis on 3 novel kinases-MBPK-55, MBPK-62 and MBPK-87. The transcription factors c-Jun, Elk, ATF2, and c-Fos that are used to assess mitogen-activated protein kinase activation were all poor substrates for these three kinases. MAPKAPK2 was also not phosphorylated. In contrast, Histone IIIS was phosphorylated by MBPK-55 and MBPK-62. These protein kinases were activated in cultured cardiac fibroblasts, H9c2 cardiac myoblasts, and Cos cells. High concentrations (0.5 to $1\;{\mu}M$) of OA were essential for the activation of the protein kinases in all of the cell types examined, whereas calyculin A [an inhibitor of protein phosphatase 1 (PP1) and PP2A], cyclosporin A (a PP2B inhibitor), and an inactive OA analog all failed to activate these kinases. The high dose of okadaic acid that is required for kinase activation was also required for phosphatase inhibition, as assessed by immunoblotting whole cell lysates with anti-phosphothreonine antibodies. A variety of chemical inhibitors, including PD98059 (MEK-specific), genistein (tyrosine kinase-specific) and Bisindolylmaleimide I (protein kinase C-specific), failed to inhibit the OA activation of these kinases. Thus, MBPK-55 and MBPK-62 are also Histone IIIS kinases that are widely expressed and specifically activated upon exposure to high OA concentrations.

• Archives of Plastic Surgery
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• v.34 no.1
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• pp.13-17
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• 2007

Purpose: To understand the pathogenesis of the disease that presents abnormally proliferated vascular endothelial cells, a model of DMH(1,2-dimethylhydrazine)-induced abnormal proliferation of HUVECs(Human Umbilical Vein Endothelial Cells) was made. We indirectly determined that Protein Kinase C(PKC) restricts the cellular proliferation and inhibits the manifestation of growth factor by using several inhibiting substances of the transmitter through our previous studies. Thereupon, we attempted to observe direct enzymatic activities of PKC and its correlation with the abnormal proliferation of vascular endothelial cells. Methods: $10^5$ HUVECs cells were applied to 6 individual well plates in three different groups; A control group cultured without treatment, a group concentrated with $0.75{\times}10^{-8}M$ DMH only, and a group treated with DMH & $5{\times}10^{-9}M$ Calphostin C, inhibitor of PKC. In analyzing the formation of intracellular PKC enzyme, protein separation was performed, and separated protein was quantitatively measured. PKC enzyme reaction was analyzed through Protein Kinase C Assay System (Promega, USA), and the results were analyzed according to Beer's law. Results: Enzymatic activity of PKC presented the highest in all reaction time of a group concentrated only with DMH, and the lowest in the control group. The group treated with DMH and the inhibitor revealed statistically lower enzymatic activity than group only with DMH in all reaction time, although higher than the control group. Conclusion: From the enzymatic aspect, most active and immediate reaction of the PKC was observed in the group concentrated with DMH only. The group treated with DMH & PKC inhibitor showed meaningful decrease. Accordingly, PKC holds a significant role in DMH-induced abnormal proliferation of vascular endothelial cells.

• Microbiology and Biotechnology Letters
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• v.21 no.1
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• pp.54-58
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• 1993

During the screening of inhibitors against protein kinase CCPKC) and the bleb formation of K562 cell induced by phorbol ester from microbial secondary metabolites, MT-2007 was purified by solvent extraction, and chromatographic techniques from Actinomycetes isolate No. 2007-18. It showed completely suppression of bleb formation of K562 cell surface induced by phorbol 12.13dibutylate at the concentration of 503.9 11M and ICso on PKC was 31.4 11M. Its structure was postulated as lasalocid A sodium salt by physico-chemical properties and UV, IR. MS, IH-NMR.

• The Korean Journal of Physiology and Pharmacology
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• v.5 no.2
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• pp.139-146
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• 2001

L-type $Ca^{2+}$ channels play an important role in regulating cytosolic $Ca^{2+}$ and thereby regulating hormone secretions in neuroendocrine cells. Since hormone secretions are also regulated by various kinds of protein kinases, we investigated the role of some kinase activators and inhibitors in the regulation of the L-type $Ca^{2+}$ channel currents in rat pituitary $GH_3$ cells using the patch-clamp technique. Phorbol 12,13-dibutyrate (PDBu), a protein kinase C (PKC) activator, and vanadate, a protein tyrosine phosphatase (PTP) inhibitor, increased the $Ba^{2+}$ current through the L-type $Ca^{2+}$ channels. In contrast, bisindolylmaleimide I (BIM I), a PKC inhibitor, and genistein, a protein tyrosine kinase (PTK) inhibitor, suppressed the $Ba^{2+}$ currents. Forskolin, an adenylate cyclase activator, and isobutyl methylxanthine (IBMX), a non-specific phosphodiesterase inhibitor, reduced $Ba^{2+}$ currents. The above results show that the L-type $Ca^{2+}$ channels are activated by PKC and PTK, and inhibited by elevation of cyclic nucleotides such as cAMP. From these results, it is suggested that the regulation of hormone secretion by various kinase activity in $GH_3$ cells may be attributable, at least in part, to their effect on L-type $Ca^{2+}$ channels.